1. Field of the Invention
The present invention relates generally to the fields of inflammation and carbohydrate and glycoprotein chemistry. More specifically, the present invention relates to novel disaccharide inhibitors of inflammation.
2. Description of the Related Art
During an inflammatory response, leukocytes move from the circulation into tissues to destroy foreign organisms and to clear damaged tissue. The first step in this process involves binding of leukocytes to cytokine activated endothelial cells lining the post-capillary venules. Activation of the endothelium causes the expression of cell surface proteins belonging to the selectin family of adhesion molecules. Vascular endothelial cells express E-selectin when induced by cytokines such as TNF-.alpha., IL-1 or LPS and P-selectin when stimulated by thrombin or histamine. Monocytes, neutrophils and lymphocytes express L-selectin. Each selectin contains a short C-terminal cytoplasmic tail, a single transmembrane domain, an EGF-like domain, and a variable number of short consensus repeats like those found in complement regulatory proteins. The amino terminal end contains a C-type lectin domain that binds carbohydrate ligands in a Ca.sup.2+ -dependent manner. The lectin binding domain recognizes specific carbohydrate structures, such as sialyl Lewis.sup.x (sLe.sup.x) present on leukocytes or sulfated ligands on the endothelium. The affinity of the interaction varies considerably and may depend on the conformation and valency of the ligand as well as differences in the carbohydrate recognition domains of the different selectins. Glycoprotein ligands identified by affinity purification contain clustered oligosaccharide chains, although monovalent carbohydrate ligands can bind as well.
Leukocytes expressing sLe.sup.x bind to activated endothelium expressing E- or P-selectins. L-selectins on the leukocytes bind to carbohydrate ligands on the endothelium such as sulfo- Lewis antigens and possibly glycosaminoglycans. The interaction results in leukocyte rolling along the endothelium. Stronger adhesion develops sequentially through chemo-attractant signaling and integrins on the leukocytes binding to intercellular adhesion molecules (ICAMS) on endothelial cells. The leukocytes subsequently extravasate across the endothelium and into the tissue.
The evidence for selectins mediating leukocyte extravasation and inflammation in vivo is compelling. Studies of selectin knock-out mice showed the absence of leukocyte rolling and a delay in neutrophil extravasation into the peritoneum. Human patients with leukocyte adhesion deficiency syndrome fail to produce sLe.sup.x and related structures and their leukocytes do not roll properly on activated endothelium. The latter study points to the importance of fucosylated ligands in selectin binding.
Sometimes the inflammatory response goes awry and destroys normal tissue. Enhanced expression of E-selectin and P-selectin occurs in the vascular endothelium of synovial tissue from rheumatoid arthritis patients. As a consequence, endothelial cells become adhesive towards monocytes, neutrophils and lymphocytes. These cells extravasate from the vessel lumen into the synovial tissue and joint fluids. The subsequent release of proteases and reactive oxygen species results in degradation of cartilage, which further exacerbates the inflammatory process and the cycle continues. Blocking the transendothelial migration of leukocytes provides a way to treat chronic inflammatory arthritis.
Selectins also play a role in the acute inflammatory response to reperfusion injury, cutaneous wounding, infection, and various models of induced lung damage. Thus, intense interest exists in developing inhibitors of selectin-carbohydrate interactions. One strategy consists of using soluble forms of selectins as competitive receptors or monoclonal antibodies directed against selectins or sLe.sup.x. These agents block neutrophil adhesion to endothelial cells in vitro and inhibit inflammation in vivo. Oligosaccharides related to Lewis X and Lewis A and inositol polyanions also inhibit leukocyte rolling on vessel endothelia and decrease inflammation.
Glycosylation inhibitors are used to study the biological function of glycoconjugates in animal cells. Plant alkaloids such a swainsonine and deoxynojiromycin derivatives block glycoprotein biosynthesis in vivo by inhibiting glycosidases involved in the maturation of Asn-linked oligosaccharides. Recently, an exocyclic epoxide derivative of glycosylceramide was shown to inhibit glycosphingolipid biosynthesis in cells, presumably by making a covalent adduct to a galactosyltransferase that acts on the ceramide intermediate. A number of other substrate-based inhibitors have been described that block glycosyltransfersases in vitro, but poor uptake has prevented them from inhibiting glycosylation in vivo.
Another class of inhibitors consist of glycosides that resemble biosynthetic intermediates involved in glycoconjugate assembly. These compounds act as substrates, produce free oligosaccharides, and divert the assembly of chains from glycoconjugates to the added acceptors. The first type of inhibitors in this class was described over twenty years ago by Okayama et al. Biochem. J. (Tokyo) 74:1069-1073 (1973). They showed that .beta.-D-xylosides stimulate the synthesis of free glycosaminoglycan (GAG) chains and competitively inhibit glycosaminoglycan formation on proteoglycan core proteins. The free glycosaminoglycan chains can have desirable biological properties as well. For example, heparan sulfate chains produced on Xyl.beta.-0-2-naphthol (naphthol-.beta.-D-xyloside) will bind to basic fibroblast growth factor, facilitating its interaction with high affinity receptors. In a similar way, GalNAc-O-benzyl stimulates mucin oligosaccharide synthesis and inhibits O-linked glycoprotein synthesis. Altering glycoprotein synthesis in HL-60 cells in this way inhibits the expression of sialyl Lewis X (sLe.sup.x) ligands and adhesion to activated endothelial cells. Acceptors consisting of two or more sugars would be desirable and more selective since many glycosyltransferases use disaccharides or larger oligosaccharides as substrates. However, poor transfer of disaccharides across cell membranes severely limits this approach.
The prior art is deficient in the lack of effective means of inhibiting the inflammatory response. The present invention fulfills this longstanding need and desire in the art.